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OBJECTIVE@#To explore the regulatory effects of GABAergic neurons in the zona incerta (ZI) on sevoflurane and propofol anesthesia.@*METHODS@#Forty-eight male C57BL/6J mice divided into 8 groups (n=6) were used in this study. In the study of sevoflurane anesthesia, chemogenetic experiment was performed in 2 groups of mice with injection of either adeno-associated virus carrying hM3Dq (hM3Dq group) or a virus carrying only mCherry (mCherry group). The optogenetic experiment was performed in another two groups of mice injected with an adeno-associated virus carrying ChR2 (ChR2 group) or GFP only (GFP group). The same experiments were also performed in mice for studying propofol anesthesia. Chemogenetics or optogenetics were used to induce the activation of GABAergic neurons in the ZI, and their regulatory effects on anesthesia induction and arousal with sevoflurane and propofol were observed; EEG monitoring was used to observe the changes in sevoflurane anesthesia maintenance after activation of the GABAergic neurons.@*RESULTS@#In sevoflurane anesthesia, the induction time of anesthesia was significantly shorter in hM3Dq group than in mCherry group (P < 0.05), and also shorter in ChR2 group than in GFP group (P < 0.01), but no significant difference was found in the awakening time between the two groups in either chemogenetic or optogenetic tests. Similar results were observed in chemogenetic and optogenetic experiments with propofol (P < 0.05 or 0.01). Photogenetic activation of the GABAergic neurons in the ZI did not cause significant changes in EEG spectrum during sevoflurane anesthesia maintenance.@*CONCLUSION@#Activation of the GABAergic neurons in the ZI promotes anesthesia induction of sevoflurane and propofol but does not affect anesthesia maintenance or awakening.
Subject(s)
Male , Animals , Mice , Mice, Inbred C57BL , Propofol/pharmacology , Sevoflurane/pharmacology , Zona Incerta , Anesthesia, General , GABAergic NeuronsABSTRACT
Manganese plays an important physiological role in the organism, and excessive manganese exposure can cause impairment of neurological and reproductive functions. Gonadotropin-releasing hormone secreted by the hypothalamus acts as an initiator to regulate reproductive functions, such as gonadal development, onset of puberty, and gonadal hormone release. But the mechanism by which manganese damages the hypothalamus leading to abnormal gonadotropin-releasing hormone release is still unclear yet. Kisspeptin, prostaglandin E2, and nitric oxide may act as stimulators to increase the release of gonadotropin-releasing hormone, while the stimulatory or inhibitory effect of γ-aminobutyric acid on the release of gonadotropin-releasing hormone is controversial. Based on current research, manganese has been less studied with Kisspeptin, and studies with prostaglandin E2, nitric oxide, and γ-aminobutyric acid mainly focused on inflammation, oxidative stress, and neurotransmitter transmission. Therefore, taking Kisspeptin, prostaglandin E2, γ-aminobutyric acid, and nitric oxide as the breakthrough points, this paper introduced the mechanism of manganese affecting the release of gonadotropin-releasing hormone in the hypothalamus through the above four pathways, and proposed that the abnormal release of gonadotropin-releasing hormone in the hypothalamus may be one of the mechanisms by which manganese regulates reproductive function, providing a new direction for the prevention and treatment of manganese-induced reproductive damage in the future.
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ObjectiveDirected differentiation of human induced pluripotent stem cells （hiPSCs） into spinal cord γ-aminobutyric acid （GABA）-ergic progenitor cells were implanted into an decellularized optical nerve （DON） bioscaffold to construct a hiPSC-derived inhibitory neural network tissue with synaptic activities. This study aimed to provide a novel stem cell-based tissue engineering product for the study and the repair of central nervous system injury. MethodsThe combination of stepwise directional induction and tissue engineering technology was applied in this study. After hiPSCs were directionally induced into human neural progenitor cells （hNPCs） in vitro， they were seeded into a DON for three-dimensional culture， allowing further differentiation into inhibitory GABAergic neurons under the specific neuronal induction environment. Transmission electron microscopy and whole cell patch clamp technique were used to detect whether the hiPSCs differentiated neurons could form synapse-like structures and whether these neurons had spontaneous inhibitory postsynaptic currents， respectively， in order to validate that the hiPSC-derived neurons would form neural networks with synaptic transmission potentials from a structural and functional perspective. ResultsThe inhibitory neurons of GABAergic phenotype were successfully induced from hiPSCs in vitro， and maintained good viability after 28 days of culture. With the transmission electron microscopy， it was observed that many cell junctions were formed between hiPSC-derived neural cells in the three-dimensional materials， some of which presented a synapse- like structure， manifested as the slight thickness of cell membrane and a small number of vesicles within one side of the cell junctions， the typical structure of a presynatic component， and focal thickness of the membrane of the other side of the cell junctions， a typical structure of a postsynaptic component. According to whole-cell patch-clamp recording， the hiPSC-derived neurons had the capability to generate action potentials and spontaneous inhibitory postsynaptic currents were recorded in this biotissue. ConclusionsThe results of this study indicated that hiPSCs can be induced to differentiate into GABAergic progenitor cells in vitro and can successfully construct iPSC-derived inhibitory neural network tissue with synaptic transmission after implanted into a DON for three-dimensional culture. This study would provide a novel neural network tissue for future research and treatment of central nervous system injury by stem cell tissue engineering technology.
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γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
Subject(s)
Glutamate Decarboxylase/genetics , Lactobacillus plantarum/genetics , Catalysis , gamma-Aminobutyric Acid , Hydrogen-Ion Concentration , Glutamic AcidABSTRACT
This study explored the effects of propofol on the activity of glutamatergic neurons in the paraventricular thalamus (PVT) and the underlying mechanisms at the molecular level using whole-cell patch-clamp techniques. Acute brain slices containing the PVT were obtained from 8 weeks old C57BL/6J mice. The electrophysiological characteristics of PVT neurons were recorded in current-clamp mode, then single-cell sequencing was used to identify neuronal types. The firing frequencies before, during, and after propofol or intralipid application were recorded as FB, FD and FW; and the membrane potentials were recorded as MPB and MPD. Picrotoxin (PTX) was used to block inhibitory gamma-aminobutyric acid type A (GABAA) receptors during the application of propofol at 10 μmol·L-1. Then, GABAA receptor-mediated spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) were recorded, and the effects of 10 μmol·L-1 propofol were investigated. The animal experiments were approved by the Medical Animal Administrative Committee of Shanghai Medical College Fudan University. The results showed that there were no significant differences in FB, FD and FW during intralipid and 2 μmol·L-1 propofol application. With propofol at 5, 10 and 20 μmol·L-1, FD decreased significantly when compared with FB, and FW increased significantly as compared with FD (P < 0.01). The inhibition degree of the three concentration groups was significantly different (P < 0.01). In addition, with propofol at 20 μmol·L-1, MPD hyperpolarized significantly (P < 0.01). In the presence of PTX, 10 μmol·L-1 propofol could not suppress the firing frequency of PVT glutamatergic neurons. Propofol at 10 μmol·L-1 prolonged the decay time of sIPSCs (P < 0.01) and mIPSCs (P < 0.05), and increased the amplitude (P < 0.01) of mIPSCs of PVT glutamatergic neurons. Together, these results indicate that propofol can inhibit the activity of PVT glutamatergic neurons in a concentration-dependent and reversible manner, and the effect is likely to be mediated by postsynaptic GABAA receptors.
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italic>γ-Aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter found in various cells in the human body. While the GABAergic system is typically associated with the nervous system, recent research has revealed that immune cells and tumor cells also express components of this system. In the tumor microenvironment (TME), GABA is secreted to act extracellularly on other cells. GABA is metabolized via the GABA shunt and is involved in the tricarboxylic acid (TCA) cycle by generating succinate, which can provide energy for tumor cells. Activation of GABA receptors (GABARs) is a major pathway through which GABA participates in the regulation of antitumor immune responses. The activation of GABA type A receptors (GABAARs) can inhibit the activation and proliferation of T cells, elicit anti-inflammatory macrophages, and promote tumor cell growth and migration, while activation of GABA type B receptors (GABABRs) is generally considered to inhibit cancer cell migration and induce cancer cell apoptosis. In general, receptor activation inhibits immune cells, but the effect on tumor cells varies. Additionally, the downregulation of the expression levels of GABA transporters (GATs) is involved in tumor progression. Although antagonists of GABA metabolism and drugs that act on GABA receptors are considered therapeutic drugs for tumors, there have been few clinical studies conducted on them.
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ObjectiveTo explore the possible mechanism of Yudian Decoction （愈癫汤） in the treatment of schizophrenia. MethodTwenty male offspring from 5 normal female 17-day-pregnant SD rats were selected as blank group. Fifteen female 17-day-pregnant SD rats were injected intraperitoneally with methyl azomethine acetate （MAM） 25 mg/kg， and the male offspring simulated the neurodevelopmental abnormality to establish a rat model of schizophrenia. Sixty successfully-modeled rats were randomly divided into 20 rats in the model group， 20 rats in the Yudian Decoction group and 20 risperidone group. After 3 days of adaptive cage feeding， the rats in the Yudian Decoction group were gavaged with 1.54 g/（kg·d） of Yudian Decoction， the risperidone group was gavaged with 0.24 mg/（kg·d） of risperidone capsule， while the blank group and the model group were gavaged with 6.7 ml/（kg·d） of distilled water， once a day， for 14 consecutive days. Sample was collected on the day after the last gavage. The expression of glutamate receptor （GluR） and γ-aminobutyric acid receptor subunit α1 （GABAARα1）-positive neurons in the hippocampus and prefrontal cortex were detected by immunofluorescence， and the positive rate was calculated； the expression of small clear proteins （PVs） in the hippocampal CA1 region and the medial prefrontal cortex was detected by immunohistochemistry； The expression of glutamic acid decarboxylase 65 （GAD65） and glutamic acid decarboxylase 67 （GAD67） proteins and mRNAs in the hippocampus and prefrontal cortex were detected by immunoblotting and reverse transcription PCR. ResultCompared with the blank group， the positive rate of GluR in hippocampal area and prefrontal cortex of rats in the model group increased， the positive rate of GABAARα1 in hippocampal area decreased， the PV optical density value in hippocampal CA1 area and medial prefrontal cortex decreased， and the expression of GAD65， GAD67 proteins and mRNA in hippocampal area and prefrontal cortex decreased （P＜0.05 or P＜0.01）. Compared with the model group， GluR positivity rate in hippocampus and prefrontal cortex of risperidone group and Yudian Decoction decreased， GABAARα1 positivity rate in hippocampus increased， PV optical density value in hippocampus CA1 area and medial prefrontal cortex increased， and GAD65， GAD67 proteins and mRNA expression in hippocampus and prefrontal cortex increased （P＜0.05 or P＜0.01）. Compared with the risperidone group， the positive rate of GluR in hippocampus and prefrontal cortex and GABAARα1 in hippocampus in the Yudian Decoction group was reduced， the PV optical density value of hippocampal CA1 area was increased， the protein and mRNA expression of GAD67 in hippocampus area was elevated， and the protein expression of GAD65 in prefrontal cortex was reduced （P＜0.05）. ConclusionYudian Decoction may improve the pathological process of schizophrenia by regulating key regulators of glutamate/γ-aminobutyric acid （Glu/GABA） metabolic balance in the hippocampus and prefrontal cortex and maintaining the balance between neuronal excitation and inhibition.
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With complex pathogenesis, myopia is a common ophthalmology disease and a major causation for visual impairment in children. For years, studies found that neurotransmitters, such as dopamine, nitric oxide, acetylcholine, γ-aminobutyric acid, 5-hydroxytryptamine, insulin and prostaglandins, are associated with children's refractive development and axial length growth. However, there are still many disagreements in their mechanisms of action. This article makes a systematic review on the roles of neurotransmitters in the pathogenesis of myopia including neurotransmitter receptors and antagonists to clarify the influence of different neurotransmitters on the occurrence and development of myopia, thus giving a comprehensive insight into its pathogenesis, building a basis for further research on the changes of neurotransmitters and providing new ideas and directions for the prevention and treatment of myopia.
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Recent studies have found that in the development of epilepsy, cyclic adenosine monophosphate response element binding protein (CREB) may cause recurrent epilepsy by inhibiting the expression of γ-aminobutyric acid, resulting in neuron damage and weakened effect of antiepileptic drug targets. Antiepileptic drugs can not control the extent or frequency of seizures, and then the patients are in a persistent state, hence the development of drug-resistant epilepsy. Therefore, the mechanism of CREB leading to drug-resistant epilepsy was reviewed in this paper, hoping to provide ideas for the treatment of drug-resistant epilepsy patients.
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Objective:To investigate the effect of aucubin on behaviors and excessive activation of astrocytic in attention deficit/hyperactivity disorder (ADHD) model mice.Methods:Twelve wild-type C57BL/6 pregnant mice (female, clean grade) were intraperitoneally administered with esketamine (15 mg/kg) to establish an ADHD model in offspring mice. The offspring mice were divided into control+ saline group, control+ aucubin group, Ketamine+ saline group and Ketamine+ aucubin group according to the nest matching principle with 15 in each group.At 14 days after birth, mice in the control+ aucubin group and Ketamine+ aucubin group were administered with aucubin (5 mg/kg, once a day) by gavage for 5 days. Mice in control+ saline group and Ketamine+ saline group were administered with equal volume of 0.9% sodium chloride solution. The offspring mice were housed with their mothers in the same cage until 21 days after birth. Twenty-one days after birth, the offspring mice were evaluated by open field test and elevated plus maze tests. Immunofluorescence assay was used to detect the expression of glutamate decarboxylase 2 (GAD2), γ- aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) in the amygdala. The morphological changes of astrocytes were quantitatively analyzed by Sholl analysis. GraphPad Prim 9.0.1 software was used for statistical analysis. The comparison of multiple groups was conducted by one-way ANOVA or Kruskal-Wallis test.Results:(1)The results of behavioral experiments showed that the total distance traveled in the open field test and the residence time in open arm of the elevated plus maze were statistically significant ( F=236.90, H=39.92, both P<0.001). The total distance ((7 044±249)mm, (22 891±2 175)mm, P<0.05) and the residence time in open arm(12.69(9.86, 17.24)s, 2.72(0.57, 3.87)s, P<0.05) of mice in Ketamine+ saline group were both higher than those in control+ saline group.The total distance((22 891±2 175)mm, (8 252±839)mm, P<0.05) and the the residence time in open arm(5.45(1.13, 10.99)s, 12.69(9.86, 17.24)s, P<0.05) of Ketamine+ aucubin group were both lower than those of Ketamine+ saline group.(2)The immunofluorescence results showed that the levels of GAD2, GABA and GFAP intensity in amygdala of mice in the four groups were statistically significant ( F=145.50, 50.08, 53.83, all P<0.05). Compared with control+ saline group, the fluorescence intensities of GAD2 ((100.00±9.60)%, (24.86±4.14)%, P<0.05) and GABA ((100.00±16.84))%, (25.48±5.70)%, P<0.05) of Ketamine+ saline group were down-regulated, and the GFAP((100.00±18.02)%, (223.80±25.85)%, P<0.05) was up-regulated. Compared with Ketamine+ saline group, the fluorescence intensities of GAD2 ((24.86±4.14)%, (56.08±6.55)%, P<0.05) and GABA((25.48±5.70)%, (52.59±15.74)%, P<0.05) in Ketamine+ aucubin group were up-regulated, but the fluorescence intensity of GFAP ((223.80±25.85)%, (157.10±22.10)%, P<0.05) was down-regulated.(3)Sholl analysis indicated that the number of the intersections between the astrocyte processes or the branches of astrocyte processes was statistically significant in the 4 groups ( F=12.47, P<0.05). Compared with control+ saline group, the number of the intersections in Ketamine+ saline group((2.07±0.48), (1.67±0.72), P<0.05) increased. While the number of the intersections in Ketamine+ aucubin group was lower than that of Ketamine+ saline group ((1.20±0.78), (2.07±0.48), P<0.05). Conclusion:Aucubin administration can alleviate ADHD-like behaviors in offspring mice, and the mechanism may be associated with the inhibition of excessive astrocytic activation.
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Objective:To investigate the effects of hydroxysafflor yellow A (HSYA) on depressive-like behavior and expression of type A γ-aminobutyric acid receptor(GABAAR)in hippocampus of chronic restraint stress model mice.Methods:The SPF grade male C57BL/6C mice were divided into Control group, HSYA group, Model group, Model + HSYA group and Model + fluoxetine group according to random number table method, with 12 mice in each group.Mice model of depression was established by chronic restraint stress.Mice in HSYA group and Model+ HSYA group were intraperitoneally injected with HSYA(20 mg/kg), mice in Model+ fluoxetine group were injected intraperitoneally with fluoxetine (10 mg/kg), and mice in Control group and Model group administered with 0.9% sodium chloride solution intraperitoneally once a day for 14 days.Then, the forced swimming test (FST) and tail suspension test (TST) were performed to evaluate the depressive-like behavior of mice, and the protein expression levels of different subtypes of GABAAR in the hippocampus of mice were determined by Western blot.SPSS 19.0 and GraphPad Prism 8.0 software were used for data statistical analysis and mapping.One-way ANOVA was used for comparison among groups, and Tukey-HSD test was used for further pairwise comparison.Results:(1) In the behavioral tests, there were significant differences in swimming immobility time of FST and tail suspension immobility time of TST among the five groups ( F=21.59, 20.81, both P<0.05). The swimming immobility time ((143.91±9.97) s) and tail suspension immobility time (( 107.00±6.54) s) in Model group were higher than those in Control group ((52.92±6.70) s, ( 43.50±5.96) s, both P<0.05). There were no significant difference in swimming immobility time and tail suspension immobility time between Model+ HSYA group ((26.17±7.69)s, ( 20.17±7.89)s) and Model+ fluoxetine group ((61.60±16.22)s, (34.14±10.74)s)(both P>0.05), but the swimming immobility time and tail suspension immobility time in these two groups were lower than those in Model group (both P<0.05). (2) The Western blot results showed that there were significant differences in the expression of GABAARβ1 and GABAARβ2 protein in hippocampus among the four groups ( F=12.21, 11.40, both P<0.05). The expression levels of GABAARβ1(45.60±10.76) and GABAARβ2 (46.27±4.82) protein in hippocampus of Model group were lower than those in Control group ((100.00±3.44), (100.00±3.26), both P<0.05). Compared to Model group, the expression of GABAARβ1 (79.91±5.00) and GABAARβ2 (79.08±5.53) protein in hippocampus of Model+ HSYA group were higher (both P<0.05). In addition, the expression of GABAARα1 and GABAARγ1 proteins in hippocampus were not significantly different among the four groups( F=0.23, 0.10, both P>0.05). Conclusion:HSYA can effectively alleviate depressive-like behavior in depression model mice, which may be related with the upregulation of GABAARβ1 and GABAARβ2 of hippocampus tissue.
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Objective:To investigate the neurobiochemical metabolites of caudate nucleus and thalamus in patients with obsessive-compulsive disorder and their relationship with obsessive-compulsive symptoms.Methods:From April 2019 to January 2022 in Beijing Anding Hospital, totally 25 untreated patients with obsessive-compulsive disorder were recruited, and 20 healthy controls matched with gender, age and educational background were recruited for the study.The maps of neurobiochemical metabolites of patients and normal controls were collected by hydrogen proton magnetic resonance spectroscopy.With bilateral caudate nucleus and thalamus as brain regions of interest.The relative concentrations of N-acetylaspartic acid (NAA), glutamic acid (Glu) and γ-aminobutyric acid (GABA) were fitted by LCModel software.At the same time, the clinical symptoms of patients were evaluated with Yale-Brown obsessive-compulsive scale (Y-BOCS) and Hamilton anxiety scale (HAMA). SPSS 20.0 software was used for statistical analysis.Independent double sample t-test was used to compare the differences of different nerve biochemical metabolite concentrations between patients with obsessive-compulsive disorders and healthy controls.Pearson correlation analysis was used to explore the correlation between biochemical metabolite concentrations and clinical symptoms. Results:The Glu concentration in the left thalamus of patients with obsessive-compulsive disorder (3.97±0.41) was higher than that of the control group (3.66±0.55)( t=-2.11, P<0.05), while the NAA concentration was (4.87±0.47)lower than that of the control group (5.15±0.44)( t=2.05, P<0.05). The GABA concentrations in the right caudate nucleus (0.50±0.18) and thalamus (0.80±0.19) were lower than those in the control group ((0.63±0.23), (0.96±0.24))( t=2.08, 2.36, both P<0.05). Pearson correlation analysis showed that the Glu concentration in the left caudate nucleus of patients with obsessive-compulsive disorder was positively correlated with the total score of Y-BOCS( r=0.46, P<0.05). Spearman correlation analysis showed that Glu concentration in the right caudate nucleus was positively correlated with the total score of HAMA in patients with obsessive-compulsive disorder ( r=0.46, P<0.05). Conclusion:NAA, Glu and GABA metabolism in caudate nucleus and thalamus are abnormal in patients with obsessive-compulsive disorder, and Glu concentration is positively correlated with the severity of obsessive-compulsive and anxiety symptoms.
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Objective:To evaluate the therapeutic effect of hydroxysafflor yellow A (HYA) on rats with tinnitus and investigate its influence on γ-aminobutyric acid (GABA) and glutamic acid (Glu) levels of inferior colliculus.Methods:The model of rats with tinnitus received an injection of sodium salicylate and "water-drinking suppression" was extablished, and then were divided into four groups with random number table method: normal group, model group, positive control (carbamazepine 5 mg/kg) and HYA (20 mg/kg) groups. Animals were intraperitoneally injected for 15 days. The recovery time of water-drinking suppression of all groups were recorded. The threshold value of auditory brainstem response (ABR) under the different frequency (4, 12, 20 and 28 kHz) in each rat was measured. The levels of GABA and Glu in inferior colliculus in rats with tinnitus were detected by LC-MS/MS.Results:Compared with the model group, the recovery time of water drinking suppression [(3.55±0.69)d vs.(1.83±0.58)d] in HYA group was significantly prolonged ( P<0.01). Compared with the model group, the threshold value of ABR under different frequency (4, 12, 20 and 28 kHz) were significantly reduced in HYA group ( P<0.01). The GABA levels [(2.25±0.26) μmol/g vs.(1.96±0.19)μmol/g] in inferior colliculus of tinnitus rats in HYA group was significantly increased ( P<0.05) while the Glu levels [(2.95±0.34)μmol/g vs.(3.71±0.39)μmol/g] were significantly decreased ( P<0.01). Conclusion:HYA treatment could relieve tinnitus symptoms induced by sodium salicylate, which might be related to the recovery of excitatory/inhibitory neurotransmitter balance.
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Objective:To observe any therapeutic effect of repeated transcranial direct current stimulation (tDCS) on rats modeling neuropathic pain and explore possible mechanisms.Methods:Forty adult male Sprague-Dawley rats were randomly divided into a normal group ( n=10), a sham operation group ( n=10), a treatment group ( n=10) and a sham treatment group ( n=10). A model of chronic constriction injury of the sciatic nerve was established in the latter two groups. Fourteen days after the modeling, the treatment group was given tDCS for 8 consecutive days, while the sham treatment group received sham stimulation, and the other 2 groups did not receive any intervention. Von Frey and hotplate tests were used to test the rats′ pain thresholds 1 day before, as well as 14 and 22 days after the surgery (i.e., 8 days after the end of the treatment). Spinal cord tissue samples were taken to detect the protein expressions of N-methyl-D-aspartic acid receptor 2B, gamma-aminobutyric acid receptor types A (GABA a-R) and B (GABA b-R) using western blotting. Results:On the 14th day after the operation the average 50% MWT and WTL values of the sham treatment and treatment groups had decreased significantly compared with the sham operation group. By the 22nd day the average 50% MWT and WTL values of the treatment group were significantly higher than those of the sham treatment group, but there was no significant change in the treatment group′s average WTL between the 21st and 22nd days. On the 22nd day after the operation the average NR2B-NMDA-R level of the sham treatment group were significantly higher than that of the sham operation group, while the average GABA a-R and GABA b-R levels were significantly lower. At the same time point the treatment group′s average NR2B-NMDA-R level had decreased significantly compared to the sham treatment group, while the average GABA a-R level had increased significantly. There was no significant difference in average GABA b-R level between the treatment group and the sham treatment group at that point. On the 22nd day there was also no significant difference in the average NR2B-NMDA-R level between the treatment group and the sham operation group. Conclusions:Repeated tDCS can effectively relieve neuropathic pain. The relief of hyperalgesia is more significant than that of mechanical allodynia. A possible mechanism may be the down-regulation of spinal NR2B-NMDA-R to normal levels and modest up-regulation of GABA a-R.
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Objective:To explore the effects of the γ-aminobutyric acid(GABA) neurons and melanin-concentrating hormone (MCH) neurons of the nucleus accumbens (NAc)-lateral hypothalamic area (LHA) neural pathway on the rewarding feeding(palatable food sweat condensed milk) in the obesity rats.Methods:Total 142 male Wistar rats of SPF grade were divided into normal diet (ND) group ( n=68) and high-fat diet induced obesity (DIO) group ( n=74) according to the principle of body mass matching. The rats in the two groups were given normal diet and high-fat diet for 8 weeks. Eight weeks later, 6 DIO rats were randomly selected to observe the nerve projection from GABA neurons in NAc to MCH neurons in LHA by fluorogold retrograde tracing combined fluorescence immunohistochemistry. And the expressions of c-Fos and MCH in LHA after ingestion of sweet condensed milk(rewarding feeding) were observed by fluorescence immunohistochemistry (6 rats in each group). GABA receptor agonist Musimol or GABA receptor antagonist Bicuculine was microinjected into the nucleus of LHA to observe the effect of GABA on rewarding food intake in ND and DIO rats ( n=8 in each group), and the changes of rewarding food intake after blocking MCH signal ( n=8 in each group). SPSS 17.0 was used for statistical analysis, two-way ANOVA and post hoc Bonferroni test were used for comparison among multiple groups, and t-test was used for comparison between two groups. Results:After 8 weeks of high-fat diet modeling, the intake of delicious food in DIO rats was significantly higher than that in ND rats((12.52±2.29) mL, (7.45±1.23) mL, t=4.778, P<0.01) after satiety.The results of fluorogold retrograde tracing combined with fluorescence immunohistochemistry showed that GABA neurons in NAc projected nerve fibers to neurons in LHA, and GABA A receptors in some neurons in LHA coexisted with MCH.The results of NAc-LHA pathway on delicious food intake showed that the interaction between rat group and drug intervention was significant( F=9.869, P<0.01). Simple effect analysis showed that the intake of delicious food after microinjection of Musimol into LHA nucleus of ND rats was significantly lower than that of microinjection normal saline ((4.25±1.38) mL, (7.29±1.49) mL, P<0.01), while the intake of delicious food after injection of Bicuculine was significantly higher than that of microinjection normal saline((10.72±2.11) mL, (7.29±1.49) mL, P<0.05). The intake of delicious food after microinjection of Musimol into LHA nucleus in DIO group was significantly lower than that of microinjection normal saline((3.51±1.77)mL, (13.68±2.95) mL, P<0.01), but there was no significant difference between microinjection Bicuculine and microinjection normal saline ((14.83±3.44) mL, (13.68±2.95) mL, P>0.05). The results of blocking MCH signal on delicious food intake showed that the interaction effect between SNAP-94847 and Bicuculine intervention was not significant ( F=1.468, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=15.880, P<0.01)and the main effect of Bicuculine intervention was significant ( F=6.930, P<0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of ND rats was significantly less than that of injection normal saline((4.78±1.72) mL, (7.63±2.77) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((6.24±2.18) mL, (4.78±1.72) mL, P>0.05). In the DIO rats, the interaction effect between SNAP-94847 and Bicuculine intervention was not significant( F=0.006, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=18.46, P<0.01) and the main effect of Bicuculine intervention was not significant ( F=2.059, P>0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of DIO rats was significantly lower than that of injection normal saline((6.89±2.11) mL, (12.19±4.36) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((8.72±2.26) mL, (6.89±2.11) mL, P>0.05). Conclusion:GABAergic signal in NAc can regulate the expression of MCH in neurons of LHA. In the DIO rats, the sensitivity of MCH neurons in LHA to satiety signal decreases and the hedonic feeding increases.
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ObjectiveTo investigate the effect of modified Renshen Wumeitang（MRWT） on the related regulatory factors of the γ-aminobutyric acid （GABA） signaling pathway in colon tissues of rats with diarrhea， and reveal the mechanism of MRWT in invigorating Qi， generating fluid， and checking diarrhea. MethodForty-eight SD immature rats were randomly divided into a blank group （n=12） and an experimental group （n=36）. The diarrhea model was induced in the experimental group by Sennae Folium combined with overstrain and improper diet for 14 days. Subsequently， the model rats were randomly divided into a model group （normal saline， 20 mL·kg-1）， a western medicine group （Medilac-Vita， 0.7 g·kg-1）， and a Chinese medicine group （MRWT， 35 g·kg-1）， with 12 rats in each group. The rats in the blank group received normal saline at 20 mL·kg-1， and those in the other groups were treated correspondingly， once a day for 7 days. The general condition， loose stool rate， and diarrhea index of the rats were observed daily. Immunohistochemistry was used to detect the optical density expression of GABA protein in the colon of rats. The content of phosphatidylinositol-3 kinase （PI3K）， protein kinase B2 （Akt2）， phosphorylated Akt （p-Akt）， and interleukin-1β （IL-1β） was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of PI3K， Akt2， and GABA type A receptor subunit β2 （GABRB2） in the colon of rats were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed worsened general condition， The difference was not statistically significant of loose stool rate and diarrhea index， increased expression of GABA protein （P<0.05）， elevated expression of PI3K， Akt2， p-Akt， and IL-1β （P<0.05， P<0.01）， and up-regulated PI3K， Akt2， and GABRB2 mRNA and protein expression （P<0.01）. Compared with the model group， the western medicine group and the Chinese medicine group showed the improved general condition， decreased loose stool rate and diarrhea index （P<0.01）， and decreased content of PI3K， Akt2， p-Akt， and IL-1β （P<0.05）. The Chinese medicine group displayed decreased mRNA expression of PI3K， Akt2， and GABRB2 （P<0.05， P<0.01） and down-regulated protein expression of GABA， PI3K， and GABRB2 （P<0.05， P<0.01）. The western medicine group exhibited down-regulated mRNA expression of PI3K，Akt2，and protein of PI3K （P<0.05）. ConclusionMRWT can regulate the GABA signaling pathway， reduce Cl- flow in intestinal epithelial cells to the intestinal lumen， and improve the imbalance of colonic fluid metabolism in the colon of diarrhea rats， thereby exerting its effects of invigorating qi， generating fluid， and checking diarrhea.
ABSTRACT
Objective:To explore the mechanism of premenstrual dysphoric disorder (PMDD) caused by liver-qi depression from the aspect of Glu-GABA metabolic pathways.Methods:Thirty-six rats with similar open field scores and regular estrus cycles were divided into blank group, model group, fluoxetine group, Shuyu capsule group, saikosaponin group and inhibitor group according to the random number table method, with 6 rats in each group. Stereotactic hippocampus surgery was performed during the first estrous cycle reception period after the estrus cycle was determined. In the non-receiving period of the third and fourth estrus cycles, the restraint model was constructed, and from the first day of the modeling, rats of the fluoxetine group were given fluoxetine capsules 2.67 mg/kg, while rats of the Shuyu capsule group and saikosaponin group were given Shuyu capsules 0.408 g/kg and saikosaponin 0.72 mg/kg once a day for 5 consecutive days. Rats in the inhibitor group were injected with 20 μl L-malic acid with 5 mmol/L concentration, which is an inhibitor of glutamate decarboxylase (GAD), in the hippocampus on the last day of modeling. After the administration, weighed the rats and carried out open field experiments. During the second and fivth estrus cycles of rats, the extracellular fluid of the hippocampus was collected by microdialysis technology, and the content of Glu and GABA in the dialysate was detected by HPLC-FLD. Results:After 5 days of administration, compared with the model group, the body weight of rats in the Shuyu capsule group, the inhibitor group and the fluoxetine group increased ( P<0.05), and the total score of the open field experiment decreased ( P<0.05); compared with the model group, during the receiving period of the five estrus cycle, the Glu level of the Shuyu capsule group and the inhibitor group decreased ( P<0.05); In the non-receiving period of the fifth estrus cycle, the Shuyu capsule group, Glu level of the fluoxetine group and the saikosaponin group increased, GABA level of Shuyu capsule group, inhibitor group and fluoxetine group decreased ( P<0.05), Glu/GABA level of Shuyu capsule group, fluoxetine group and inhibitor group (1.49 ± 0.13, 1.32 ± 0.33, 3.92 ± 0.79 vs. 0.35 ± 0.48) was higher than that of the model group ( P<0.05). Conclusion:The therapeutic mechanism of Shuyu capsule in the treatment of PMDD caused by liver Qi depression rats may be ascribed to inhibiting GAD from Glu-GABA metabolic pathway.
ABSTRACT
To investigate the effect of electro-acupuncture therapy on limb spasm and excitability of motor neurons in stroke rats. Ischemic stroke model was induced with middle cerebral artery embolization in SD rats. Thirty-three modeled rats were randomly divided into model group, electro-acupuncture group, and baclofen group with 11 rats in each group, and another 10 rats were taken as sham operation group. The electro-acupuncture group and the baclofen group were treated with electro-acupuncture and baclofen tablets respectively. The model group and the sham operation group had no intervention. The neural function was evaluated with Bederson's scale and balance beam test; the muscle tension was measured with electrophysiography; the pathological changes of brain tissue was examined with HE staining; the content of glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rat cerebral cortex was analyze with enzyme linked immunosorbent assay (ELISA) method, the expression of metabotropic glutamate receptor 1a () and γ-aminobutyric acid type B receptor subunit 1 () mRNA were detected with RT-qPCR. Compared with the model group, the neurological function scores of the electro-acupuncture group and the baclofen group showed a downward trend at d7 after operation (all >0.05), and the neurological function scores of the electro-acupuncture group and the baclofen group were significantly decreased at d12 after the operation (all 0.05). Compared with the model group, the electrophysiological results of the electro-acupuncture group and baclofen group were significantly increased after operation (all <0.05). The results of HE staining showed that there was no cell edema and degeneration in the sham operation group, no pyknosis of the nucleus, and no bleeding in the interstitium. Cell edema and degeneration and mesenchymal congestion appeared in the model group. Compared with the model group, the cytoplasmic edema and degeneration and the interstitial bleeding in the electroacupuncture group and the baclofen group were reduced. Compared with sham operation group, the Glu content and the relative expression of mRNA was increased in the model group, electro-acupuncture group and baclofen group, while the GABA content and the relative expression of mRNA decreased (all <0.05). Compared with model group, the Glu content and the relative expression of mRNA in the electro-acupuncture group and baclofen group decreased, and the GABA content and relative expression of mRNA increased (all <0.05). Electro-acupuncture may improve limb spasm after stroke through regulating the expression of Glu and GABA in the cerebral cortex and the excitability of motor neurons in rats.
Subject(s)
Animals , Rats , Acupuncture Therapy , Motor Neurons , Rats, Sprague-Dawley , Spasm , Stroke/therapyABSTRACT
Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.
Subject(s)
Aminobutyrates , Escherichia coli/genetics , Leucine Dehydrogenase/genetics , Threonine DehydrataseABSTRACT
OBJECTIVES@#To explore the effect of etomidate on the neuronal activity of ventral thalamic reuniens nucleus and the underlying mechanisms.@*METHODS@#Whole-cell patch clamp method was used to explore the effect of etomidate on the activity of ventral thalamic reuniens neurons in the acute brain slices obtained from 4-5 weeks old C57BL/6J mice. The electrophysiological characteristics of ventral thalamic reuniens neurons were recorded in the current clamp mode, and then the effects of etomidate (0.5, 2.0, 8.0 μmol/L etomidate groups) and intralipid (intralipid group) on the discharge frequency and membrane potential of ventral thalamic reuniens neurons were recorded. During the experiment, the ventral thalamic reuniens neuron firing rates (RNFRs) were recorded as F@*RESULTS@#In the intralipid group, there was no significant difference among the F@*CONCLUSIONS@#Etomidate can inhibit the activity of ventral thalamic reuniens neurons in concentration-dependent manner, and which is reversible. Etomidate with sub-anesthetic concentration inhibits the activity of ventral thalamic reuniens neurons via targeting the GABA